Hoechst 33342 Solution — 561908 - BD Biosciences

1 BD Pharmingen Bioimaging Certified ReagentTechnical Data SheetHoechst 33342 SolutionProduct InformationMaterial Number: 561908 Size: 1 mg/mlDescriptionThe BD Pharmingen Hoechst 33342 Solution is a reagent for the fluorescent staining of DNA and nuclei in live or fixed cells. Hoechst 33342 is a bisbenzimidazole dye with high specificity for binding to double-stranded DNA (preferentially binds to A-T base pairs). This dye is very useful to label double-stranded DNA and thus to visualize nuclei. Hoechst 33342 can be excited at ~355 nm by a UV light source (eg, UV laser beam or a mercury arc-lamp). It emits blue fluorescence light around an emission maximum at 461 nm when bound to DNA. Since the Hoechst 33342 dye is specific for DNA binding, ribonuclease treatment is not needed to avoid nonspecific RNA staining. In addition to its use in fluorescence microscopy and image analysis, Hoechst 33342 is commonly used for flow cytometric applications, such as cell cycle analysis and stem cell side population immunofluorescence analysis of cell cycle of HeLa cells (Left Panel).

2 HeLa cells (ATCC CCL-2) were stained with BD Pharmingen Cell Cycle Kit (Cat. No. 558662) with Alexa Fluor 488 Mouse anti-BrdU ( pseudo-colored green), Alexa Fluor 647 Rat anti-Histone H3 (pS28) (pseudo-colored red), and Hoechst 33342 (Cat. pseudo-colored blue). Co-staining of Hoechst 33342 and Histone H3 (pS28) appears pink. The confocal image was captured using a 20x ( NA) objective with the BD Pathway 435 Bioimaging Analyzer and merged using BD Attovision cytometric analysis of HeLa cell DNA content (Right panel). HeLa cells in log growth were dissociated from the growth medium using Cell Dissociation Buffer (Life Technologies) and resuspended in complete medium containing 10 g/mL Hoechst 33342 for 60 minutes at 37 C. Cells were pelleted by centrifugation, Hoechst -containing medium was removed, and cells were resuspend in PBS and analyzed for DNA content. Data was acquired on a BD LSRF ortessa cell analyzer system. DNA content histogram was deconvoluted into G0/G1, S, and G2/M populations by FlowJo software (TreeStar).

3 Preparation and StorageStore undiluted at 4 C and protected from prolonged exposure to light. Do not NotesApplicationBioimagingTested During DevelopmentImmunofluorescenceTested During DevelopmentIntracellular staining (flow cytometry)Tested During DevelopmentRecommended Assay Procedure: Immunofluorescent Staining of Live Cells for Nuclear Hoechst 33342 Solution to 5 - 10 g/mL in complete medium immediately prior to Hoechst 33342 Solution to each sample and incubate at 37 C for 30 - 60 minutes. The stain time required is cell type Hoechst Solution from cells at the end of the incubation period and add BD Pharmingen Stain Buffer (FBS) (Cat. No. 554656) or 1 PBS. Cells may also be analyzed without washing, but this may increase background from unbound to imaging. 561908 Rev. 2 Page 1 of 2 Immunofluorescent Staining of Fixed Cells for Nuclear and permeabilize cells as desired. Hoechst 33342 Solution to 2 g/ml in 1 PBS immediately prior to use. 2ug/ml Hoechst 33342 Solution to each sample at least 15 minutes before to of Live Cells for DNA Content Analysis by Flow a single cell cells at 1x10^6 cells/mL or less in complete medium containing 5 - 10 g/mL Hoechst 33342 .

4 Note: Alternatively, Hoechst 33342 may be added directly to culture medium without pelleting if the culture cell density does not exceed 1x10^6 at 37 C for 30 - 60 optimal cell density, concentration of Hoechst 33342 , and stain time for DNA content analysis may vary by cell type. Assay conditions should be optimized in early experiments for best results. cells by centrifugation and aspirate medium containing Hoechst cells in BD Pharmingen Stain Buffer (FBS) or 1 PBS and proceed to analysis by flow of Fixed Cells for DNA Content Analysis by Flow a single cell cells on ice for 30 minutes with 70 - 80% ice-cold fixation typically provides the most resolved histograms. However, this reagent has also been successfully used for DNA content analysis with the Transcription Factor Buffer Set (Cat. No. 562574) or BD Cytofix Fixation Buffer (Cat. No. 554655) and BD Phosflow Perm III (Cat. No. 558050) protocol. cells once with BD Pharmingen Stain Buffer (FBS). Hoechst 33342 Solution to 1 - 5 g/mL in BD Pharmingen Stain Buffer (FBS) or 1 PBS immediately prior to cells for 15 minutes at a cell density of 1x10^6 cells/mL.

5 No wash is necessary prior to optimal cell density and concentration of Hoechst 33342 for DNA content analysis may vary by cell type. Assay conditions should be optimized in early experiments for best to analysis by flow cytometry. This product is also available as a component of the Cell Cycle Kit (Cat. No. 558662). Please see the kit's Technical Data Sheet for a detailed protocol for the use of Hoechst Dye 333342 in conjunction with immunofluorescent staining of plated Companion ProductsCatalog NumberNameCloneSize558662 Cell Cycle Kit100 mg(none)554655 Fixation Buffer100 mL(none)562574 Transcription Factor Buffer Set100 Tests(none)558050 Perm Buffer III125 mL(none)554656 Stain Buffer (FBS)500 mL(none)Product NoticesPlease refer to for technical protocols. 1. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.

6 2. Since applications vary, each investigator should titrate the reagent to obtain optimal results. 3. Alexa Fluor is a registered trademark of Molecular Probes, Inc., Eugene, OR. 4. FlowJo is a trademark of Tree Star Inc. 5. ReferencesCrissman HA, Steinkamp JA. Multivariate cell analysis. Techniques for correlated measurements of DNA and other cellular constituents. In: Gray JW, Darzynkiewicz Z, ed. Techniques in Cell Cycle Analysis. Clifton, NJ: Humana Press; 1987:163-206. (Methodology: Flow cytometry)M ller W, Gautier F. Interactions of heteroaromatic compounds with nucleic acids. Eur J Biochem. 1975; 54(2):385-394. (Methodology)Shapiro HM. Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry. 1981; 2(3):143-150. (Methodology: Flow cytometry) 561908 Rev. 2 Page 2 of 2